Introduction

Prostate cancer is the most common cancer in men and the leading cause of male cancer death, after lung cancer. It is predominantly a disease of elderly men, its incidence increasing steeply in the seventh decade of life (Quinn and Babb 2002). Similar to other malignancies, autocrine and paracrine growth factors and its receptor interactions leads to the deregulated prostate cancer cell growth, i.e., androgen independent prostate cancer (Gioeli et al. 1999; Agarwal 2000). Since, in advanced stage, prostate cancer growth and development become androgen-independent that renders androgen ablation therapy ineffective (Aquilina et al. 1999); so it is necessary to control prostate cancer through chemoprevention and intervention of new therapies. Several epidemiological and laboratory studies have shown that many vegetables, fruits and grains as well as phytochemicals offer significant protection against various cancers including prostate cancer (see Taraphdar et al. 2001). Quercetin, a flavonoid commonly present in many vegetables has been shown to induce apoptosis in many tumor cell lines (Csokay et al. 1997). It can interact with a broad range of enzymes specifically receptor kinases, protein kinase C, cyclin-dependent kinases (Cdks) and also with MEK-ERK signaling (Ferriola et al. 1989; Casagrande and Darbon 2001; Nguyen et al. 2004).

Despite a large amount on the antiproliferative and proapoptotic properties, the exact mechanism by which quercetin exerts its effects on prostate cancer is unknown. Knowles et al. (2000) demonstrated the dose-dependent decrease in proliferation of PC-3 cells by quercetin. In addition, it has been shown that quercetin inhibits IGF-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in in vitro (Wang et al. 2003). However, there is no clear study on the quercetin role on cell cycle regulation of human prostate cancer cells. Cell proliferation involves the activation of several cell cycle regulators including cyclin-dependent kinases (Cdks), cyclins, CDK-inhibitors, Retinoblastoma protein (pRb) and its related proteins such as pRb/p107 and pRb2/p130, subsequently resulting in activation of E2F transcription factor family and commanding cell growth, proliferation and cell survival (Morgan 1995; Mittnacht 1998; Dyson 1998; Grana and Reddy 1999). Hypophosphorylated pRb have been shown to inhibit cell proliferation by sequestering E2F transcription factors (Howard et al. 2000).

Taken together, developing strategies that could halt cell cycle progression by targeting Cdks or pRb protein could be useful for the prevention and/or intervention of the advance stage of androgen independent prostate cancer. In order to study the molecular mechanism involved in the anticancer effect of quercetin on prostate cancer, we focused our attention on the cell cycle progression and apoptosis. Since, in advance prostate carcinoma PC-3 cells, p53 gene is mutated and their protein is non-functional, we hypothesized that Cdc2/Cdk-1 and pRb may be the likely tumor suppressor target for quercetin in modulating the cell cycle progression.

Materials and methods

Minimum essential medium (MEM), fetal bovine serum (FBS), trypan blue and quercetin were purchased from Sigma Chemical Co., USA. Thymidine [3 H] was purchased from BRIT, Mumbai, India. Other chemicals were obtained from Sisco Research Laboratories (SRL), India. All the chemicals used were extra pure and are culture grade. The androgen-independent prostatic carcinoma PC-3 cell line was obtained from National Centre for Cell Science, Pune, India. Quercetin was dissolved in dimethyl sulfoxide (DMSO). Doses were selected based on our previous study (Vijayababu et al. 2004). DMSO in culture media never exceeded 0.1% (v/v), the concentration known not to affect the cell proliferation. Cell viability was tested by trypan blue exclusion method. PC-3 cells were plated at 1×105 cells per well in 12-well culture plates containing MEM with 5% FBS. The growth inhibitory effect of quercetin was studied using Thymidine [3 H] incorporation.

Cell proliferation by thymidine uptake

Cell proliferation was assessed by Thymidine incorporation method (Martin and Pattison 2000). During the final fourth hour of quercetin treatment, 1 μCi/well [3H] thymidine-containing medium was added and incubated. Monolayers were rinsed twice with ice-cold saline and fixed with 1 ml/well ice-cold methanol–acetic acid mixture at 4°C for a minimum of 2 h. Cells were solubilized in 0.5 ml of SDS and 250 μl of each lysate was mixed with the scintillation fluid, before counting. The samples were counted using 1409 Wallac DSA liquid scintillation counter.

Flowcytometry analysis

PC-3 cells were plated at a density of 1×l06 cells/petri dish. Quercetin-treated cells were trypsinized and removed at the indicated time (i.e., 24 h) from culture dishes by centrifugation. After washing with ice-cold PBS, cells were suspended in about 0.5 ml of PBS, 0.8 ml of solution containing 1.5% Triton X-100, 20 μg/ml of RNase, and 50 μg/ml of propidium iodide were added, and the cells were kept at 4°C for 30 min. Cell cycle analysis was performed using a Beckman Vantage flow cytometer, and quantitation of cell cycle distribution was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA, USA).

Western blotting

Cells were plated on 75 cm2-flasks at density of 2×106 cells per flask, allowed to attach overnight and then exposed to quercetin for 24 h. Control cells were exposed to DMSO containing MEM medium for 24 h. After the cells were washed three times with ice-cold PBS, then total proteins were extracted by adding 200 μl of cold lysis buffer (50 mM Tris–HCl, pH 7.4; 1 mM sodium fluoride (NaF); 150 mM NaCl; 1 mM EGTA; 1 mM phenylmethylsulphonyl fluoride (PMSF) and 10 mg/ml leupeptin) to the cell pellets. After 30 min on ice, the cell debris was pelleted by centrifugation at 10,000×g for 30 min at 4°C. The total protein was quantified by Lowry’ s method (1951). The samples (50 μg of total protein) were mixed with 5X sample buffer, containing 0.3 M Tris–HCl (pH 6.8), 25% 2-mercaptoethanol, 12% sodium dodecyl sulphate (SDS), 25 mM EDTA, 20% glycerol, and 0.1% bromophenol blue. The mixtures were boiled at 95°C for 5 min, and subjected to polyacrylamide gel electrophoresis (SDS-PAGE) at a constant current of 20 mA. Following electrophoresis, proteins on the gel were electro-transferred onto PVDF membrane (Millipore, Bedford, MA, USA) in transfer buffer composed of 25 mM Tris–HCl (pH 8.9), 192 mM glycine and 20% methanol. The membranes were blocked with 20 mM Tris–HCl (pH 7.4), 125 mM NaCl, 0.2% Tween 20, 1% bovine serum albumin (BSA), and 0.1% sodium azide. The membranes were then immunoblotted with human pRb (Nova Castra, UK, generous gift from Prof. Franca Esposito, Universita’ di Napoli, Italy), pRb2/p130, Cdc2/Cdk-1, p21/Cip1, cyclin A, cyclin B1 (Calbiochem, USA), Bcl-2, Bax, Bcl-XL(generous gift from Dr. Ion V. Deaciuc, University of Louisville, Kentucky, USA) and β-actin antibodies. After washing, the membranes were incubated with horseradish peroxidase-labeled antimouse Rabbit IgG antibody at a dilution of 1:1,000. The bands were developed using ECL kit (Perkin Elmer).

TUNEL

DNA strand breaks in apoptotic cells were measured by terminal deoxynucleotidyl transferase mediated biotinylated UTP nick end-labeling (TUNEL) using an in situ cell death detection kit from Roche Molecular Biochemicals, Germany. Treated cells were harvested and fixed with 4% paraformaldehyde solution and incubated in a 0.1% Triton permeabilization solution on ice according to the manufacturer’s instructions. Cells incubated with the solution without the terminal transferase was used as a negative control.

Statistical analysis

The data were analyzed using the SPSS 7.5 Windows Students version software. For all the measurements, one-way ANOVA followed by Student’s Newman Keuls (SNK) test was used to assess the statistical significance of difference between control and quercetin-treated. A statistically significant difference was considered at the level of P<0.05.

Results

Quercetin inhibits growth of human prostate carcinoma PC-3 cells

PC-3 cells showed a significant decrease in thymidine [3H] uptake. Figure 1 shows the kinetics of proliferation up to 0–72 h quercetin treatments, during which the thymidine uptake was decreased between threefold and fourfold. Time response data demonstrate 50% growth inhibition was observed at 100 μM for 24 h.

Fig. 1
figure 1

Effect of quercetin on PC-3 cell proliferation. 3×104 cells were plated in 24-well plates. After reaching 70–80% confluence, in the presence of 5% FBS, cells were treated with vehicle or with various concentrations of quercetin (25, 50, 75, and 100 μM) for 24, 48 and 72 h. Proliferation of cells was quantitated by [3 H] thymidine incorporation. Experiments were performed in triplicate, values represents % of cpm and SD was less than 10%. a Represents the level of statistical significance at P<0.05 using SNK test between control and quercetin treatment groups

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Quercetin induces peak at Sub G1 in PC-3 cells

Consistent with its effect on cell growth inhibition, quercetin induced significant (P<0.05) G2-M arrest in PC-3 cells (Fig. 2) quercetin treatment for 24 h resulted in accumulation of 9–11% of cells in sub-G1 phase compared with control. The observed increase in sub G1 cell population was accompanied by increase in the number of apoptotic cells.

Fig. 2
figure 2

Effects of quercetin on cell cycle distribution in PC-3 cells. PC-3 cells were treated with quercetin at doses of 50 and 100 μM for 24 h in a 25 cm2 flasks. After treatment 1×106 cells were trypsinized and suspended in 1 ml of fluorochrome solution (50 μg/ml propidium iodide, 20 μg/ml RNase A, 1.5% Triton X-100) for at least 1 h in the dark at 4°C. Cell cycle analysis was performed using a Beckman Vantage flow cytometer, and quantitation of cell cycle distribution was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). Significant decrease in the proportions of cells in G, S and also G2-M-phases, were evident after exposure to quercetin for 24 h. A concomitant increase of cells in Sub G1-phase was observed in quercetin treated cells, indicating induction of apoptosis (Table 1)

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Phase

Control

50 μ M

100 μ M

Sub G1

 

9.8+0.8*

11+0.82*

G1-S

80.2+6.8

68.6+6.1*

64+6.2*

G2-M

19.8+1.1

21.6+1.2

25+1.6*

Quercetin induces cell cycle arrest at G2-M phase

Based on the data showing that quercetin induces strong G2-M arrest in PC-3 cells, we assessed the effect of quercetin in the cell cycle regulatory modules that play important roles in G2-M phase of cell cycle progression. Since PC-3 cells do not have functional p53 protein, our focus was to assess the protein expression of Cdc2/Cdk-1, the down-regulated level of which have been shown to be associated with G2-M arrest. Western blot analysis showed that quercetin treatment of PC-3 cells strongly induced the expression of p21/Cip1 (Fig. 3). Perturbations in cell cycle regulation have been demonstrated as one of the most common features in cancer cells. These alterations are generally associated with uncontrolled cell growth involving a lack of p21/Cip1 or loss of its function and increased expression of Cdks and cyclins. Accordingly, we next assessed the effect of quercetin on the expression of Cdks and cyclins involved in G2-M phase transtition of cell cycle progression. As shown in Fig. 3, quercetin treatment of PC-3 cells showed decreased the expressions of Cdc2/Cdk-1, cyclin B1 and no change in cyclin A protein.

Fig. 3
figure 3

Western blot analysis of cell cycle regulatory proteins p21/Cip1, cyclin-dependent kinase-1 (Cdc2/Cdk-1), pRb, pRb2/p130, cyclin A, cyclin B1 and β-actin expression in PC-3 cells treated for 24 h with quercetin. Equal volumes of whole-cell extracts containing 50 μg of protein were separated and electrophoretically blotted

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Quercetin increases hypophosphorylated form of pRb

Since quercetin showed strong decrease in the expression of Cdc 2/Cdk-1 and its partner cyclin B1, our next focus was to investigate the phosphorylation status of pRb protein. Quercetin treatment of PC-3 cells at 50 and 100 μM for 24 h, showed increase in hypophosphorylated levels of pRb in a dose-dependent manner (Fig. 3). But there was no change in the pRb2/p130 protein level in quercetin treated cells. These results suggest that growth inhibiting effect of quercetin in Cdk-1 expression followed by modulating further down stream targets such as pRb.

Quercetin causes apoptosis in PC-3 cells

p53-dependent and independent pathways are known to exist as major apoptosis pathways. However, the p53 protein was not detected in this cell line. Not only p53, but also proteins of the Bcl-2 family are important in regulation of apoptosis. Bax is known as a pro-apoptotic protein and it forms homo-or hetero dimers with the anti-apoptotic proteins of Bcl-2 and Bcl-XL. In this study, the level of Bax in the PC-3 cells incubated with quercetin increased markedly in a dose dependent manner (Fig. 4). The level of expression of Bcl-2 and Bcl-XL in the quercetin treated cells were also significantly decreased. Quantitative apoptotic cell death was performed to confirm the induction of apoptosis. Quercetin-caused apoptotic death of PC-3 cells was analyzed by TUNEL assay, which showed that 50 and 100 μM treatment of quercetin for 24 h significantly (P<0.05) increased the percentage of apoptotic cells up to tenfold compared with that of control (Fig. 5). To further confirm the apoptotic effect of quercetin, western blot analysis was performed to analyze caspase-3 (active fragment). As shown in Fig. 6, quercetin increased the levels of caspase-3, suggesting the possible involvement of caspase-3 activation as one of the possible mechanism of apoptosis induction.

Fig. 4
figure 4

Effects of quercetin on Bcl-2, Bcl-XL and Bax levels in prostate cancer cell line (PC-3). PC-3 cells were treated with indicated concentrations of quercetin for 24 h. Cell lysates were analyzed by Western blotting. Blots were incubated with anti-Bcl-2, anti-Bcl-xL and Bax antibodies and then incubated with anti mouse secondary antibody. Bands were developed with ECL kit (Perkin Elmer, USA)

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Fig. 5
figure 5

Effect of quercetin on induction of apoptosis in PC-3 cells. The percentage of apoptotic cells was determined and summarized, generation of free 3′-OH DNA fragments was determined using TUNEL assay. Experiments were performed in triplicate, values represents the percentage of apoptotic cells and SD was less than 10%. a Represents the level of statistical significance at P<0.05 using SNK test between control and quercetin cells

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Fig. 6
figure 6

Effect of quercetin on the expression of caspase-3 in prostate cancer cell line. PC-3 cells were treated with indicated concentration of quercetin. After 24 h incubation, cells were lysed and equal proteins were resolved on SDS polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the antibodies against caspase-3 and β-actin. Proteins were visualized using ECL detection system. β-actin was used as an internal control

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Discussion

The mammalian cell cycle is regulated by complex machinery, in which Cdks, CDKIs and cyclins play essential roles (see Morgan 1995). CDKIs are tumor suppressor proteins that down regulate the cell cycle progression by binding with active Cdk-cyclin complexes and thereby inhibiting their kinase activities (Pavletich 1998). The important CDKIs include p21/Cip1, a universal inhibitor of Cdks whose expression is mainly regulated by the p53 tumor suppressor protein (Xiong et al. 1993). The increased expression of M cyclins in cancer cells provide them an uncontrolled growth advantage because most of these cells either lack p21 or possess non-functional p21 or have low expression of p21 (Malumbres and Barbacid 2001; Gali-Muhtasib and Bakkar 2002). Previously it was reported that quercetin downregulated the androgen receptor expression by upregulating the levels of c-jun in two androgen dependent prostate cancer cells (LNCaP and LAPC-4) and brought inhibition of proliferation of prostate cancer cells (Yuan et al. 2004). There are few reports available on the anticancer effects of quercetin on androgen-independent prostate cancer cells (PC-3) (Knowles et al. 2000; Wang et al. 2003), but the exact mechanism by which quercetin exerts its effects on this cell line is unknown.

The novel finding of the present study is that quercetin strongly induced p21 and decreased the Cdc2/Cdk-1 and cyclin B1 expression followed by an increase in hypophosphorylated levels of pRb protein. These molecular effects of quercetin could be one of the possible underlying mechanisms that resulted in inhibiting cell proliferation and G2-M arrest in PC-3 cell cycle progression. When cell cycle phase distributions are compared with alterations in cell cycle attributed as one of the major causes of quercetin-induced G2-M arrest and cell growth inhibition. This finding is not consistent with earlier reports in which quercetin has been shown to induce p21/Cip1 and G1 arrest in cancer cells (Choi et al. 2001). However, it should also be noted that quercetin induces G2-M arrest in human breast cancer cells without altering Cdc2/Cdk-1 but upregulated the cyclin B1 protein expression (Balabhadrapathruni et al. 2000), suggesting a different mechanism of cell cycle arrest by quercetin and that could be most likely due to dissimilar origin of the cells.

pRb is under phosphorylated in G0 and early G1. In late G1, the protein becomes phosphorlyated at restriction point and phosphorylation increases in S phases and at the G2M phase transition (Buchkovich et al. 1989). This pRb is a nuclear phospho-protein and is regulated in a cell cycle-dependent manner by phosphorylation, and are critical targets for inactivation by transforming oncoproteins (Howard et al. 2000; Paggi et al.1996, 2003). This protein bind to and modulate the activity of E2F family of transcription factors that induce the transcription of genes needed for the cell cycle progression through G1, S, G2 and M phases (Ewen 1994). With a closer look at pRb phosphorylation at different phases of the cell cycle, we can argue that cyclin A and cyclin B1/cdc 2 might be responsible for G2M phosphorylation of pRb (Lees et al. 1991; Lin et al. 1991). Since, quercetin treated cells showed a strong decrease in protein expression of Cdc2/Cdk-1, the level of phosphorylation status of pRb was significantly decreased, but there was no change in the pRb2/p130 expression. This suggests that quercetin inhibited the phosphorylation of pRb through inhibition of Cdc2/Cdk-1 kinase. Our data also suggest that quercetin treatment caused an up-regulation of p21/Cip1 protein by p53-independent pathway, as PC-3 cells lack functional p53.

It is well known that many factors are involved in the apoptosis pathway. To clarify the mechanisms of induction of apoptosis by quercetin, we investigated the expression of apoptosis-inhibiting and apoptosis-promoting proteins in PC-3 cells. Proteins of the Bcl-2 family are known to regulate promotion and inhibition of apoptosis (Srivastava et al. 1998). Members of the Bcl-2 family are expressed in prostate cancer cells (see Catz and Johnson 2003). The Bax protein is known to be an apoptosis induction factor because of the formation of homo- and hetero dimers with the apoptosis suppressing factors Bcl-2 and Bcl-XL (Li et al. 2001). In the present study, quercetin induced a strong expression of Bax protein, on the other hand, the level of expressions of Bcl-2 and Bcl-XL decreased in a dose dependent manner. Furthermore, quercetin caused apoptotic cell death in human prostate cancer cells that was evidenced by presence of active fragment of caspase-3.

In summary, quercetin was found to inhibit the proliferation of PC-3 cells through induction of cell cycle arrest and apoptosis. The mechanism of induction of apoptosis by quercetin are thought to differ, and are p53 independent. Our findings suggest that quercetin downregulates expression of Bcl-2 and Bcl-XL and upregulates expression of Bax and thereby induces apoptosis in PC-3 cells. In addition, quercetin also caused a significant decrease in Cdc2/Cdk-1 and cyclin B1 protein expressions and increased hypophosphoryalted level of pRb and this may be attributed to decreased expression of growth responsive genes and subsequent growth inhibition of PC-3 cells. The pRb protein was found to be one of the targets for the quercetin-induced cell cycle arrest. Accordingly, the observed biological effects of quercetin in PC-3 cells and their associated mechanisms are encouraging, and need more detailed study to justify the modulation of these molecular event towards anticancer effect of quercetin against human prostate cancer.